Method for nerve growth induction

ABSTRACT

A composition comprising nerve growth factor and 2-amino-1,1,3-tricyano-1-propene useful for the induction, stimulation, and maintenance of nerve growth, and methods of potentiating choline O-acetyltransferase and tyrosine hydroxylase by 2-amino-1,1,3-tricyano-1-propene are disclosed.

This is a division of application Ser. No. 253,167 filed Oct. 4, 1988,now U.S. Pat. No. 5,023,238.

INTRODUCTION

The present invention relates to nerve growth induction, stimulation,and maintenance, and enzyme potentiation. More particularly, the presentinvention relates to a composition comprising nerve growth factor and2-amino-1,1,3-tricyano-1-propene and a method of inducing, stimulating,and maintaining nerve growth therewith, and methods of potentiatingcholine-O-acetyltransferase and tyrosine hydroxylase by means of2-amino-1,1,3-tricyano-1-propene.

BACKGROUND OF THE INVENTION

Nerve growth plays a major role in the development of host nervoussystems, as well as the survival and regeneration of component nervecells subject to damage or destruction by injury or disease, such ascognitive disorders associated with dementia.

Nerve growth factor, a polypeptide, induces nerve growth in hosts (forreviews on nerve growth factor, see L. A. Green and E. M. Shooter, Ann.Rev. Neurosci., 3, 353 (1980) and B. A. Yanker and E. M. Shooter, Ann.Rev. Biochem., 51, 845 (1982)). 2-Amino-1,1,3-tricyano-1-propene, adimer of malnonitrile, also promotes nerve growth in host systems (see,for example, R. T. Houlihan and J. P. DaVanzo, Experimental Neurology,10, 183 (1964). It has now been found that nerve growth factor incombination with 2-amino-1,1,3-tricyano-1-propene synergisticallyinduces, stimulates, and maintains nerve growth, thereby rendering thecombination more effective than either component in restoring nervefunction diminished by injuries or degenerative conditions, e.g.,Alzheimer's disease (see F. Hefti and W. J. Weiner, Annals of Neurology,20, 275 (1986).

Cholinergic and adrenergic defects are also implicated in nervedegenerative disorders (see, for example, K. L. Davis and R. C. Mohs,The New England Journal of Medicine, 315, 1286 (1986). It has now alsobeen found that 2-amino-1,1,3-tricyano-1-propene potentiatescholine-O-acetyltransferse and tyrosine hydroxylase, thereby augmentingits nerve growth restorative properties and usefulness in nervedegenerative conditions including, e.g., Parkinson's disease, S. H.Appel, Ann. Neurol., 10, 499 (1981).

DESCRIPTION OF THE INVENTION

The present invention relates to a composition comprising nerve growthfactor and 2-amino-1,1,3-tricyano-1-propene useful for the induction,stimulation, and maintenance of nerve growth in hosts. The presentinvention also relates to the potentiation ofcholine-O-acetyltransferase and tyrosine hydroxylase by2-amino-1,1,3-tricyano-1-propene.

As used throughout the specification and appended claims, the phase"inducing nerve growth" refers to the production of nerve cells fromnon-neuritic cells; the phrase "stimulating nerve growth" refers to theenhanced production of nerve cells from neuritic cells; the phrase"maintaining nerve growth" refers to the protection or continuingexistence of nerve cells; the term "potentiating" refers to theenhancement of the effects of an agent by another agent so that thetotal effect is greater than the sum of the effects of either agent; theexpression "cholinergic nerves" refers to nerves which liberateacetylcholine at a synapse; and the expression "adrenergic nerves "refers to nerves which liberate catecholamines.

2-Amino-1,1,3-tricyano-1-propene is prepared by the methods described inU.S. Pat. No. 2,719,861, issued Oct. 4, 1955.

Nerve growth factor is isolated by the processes reported in S. Varon,et al., Biochem., 6, 2202 (1967). Included among nerve growth factorsare those derived from fish, reptiles, avaian species, and mammals suchas mice and rabbits. The male mouse submaxillary gland is a particularlyabundant source of nerve growth factor.

Administration of a composition of nerve growth factor and2-amino-1,1,3-tricyano-1-propene to a host, a mammal, for example, amouse or a rabbit induces, stimulates, and maintains nerve growth withinnervous systems. Among nervous systems there may be mentioned thecentral, peripheral, and autonomic nervous systems. Representativenerves of the central nervous systems are cholinergic and adrenergicnerves; representative nerves of the peripheral nervous system are thesciatic, ulnar, radials, and median nerves; and representative nerves ofthe autonamic nervous system are the vagus, facial, glosso-pharyngeal,and spanchnic nerves.

The nerve growth induction, stimulation, and maintenance effects of thecomposition of nerve growth factor and 2-amino-1,1,3-tricyano-1-propeneare demonstrated as follows:

Rat adrenal pheochromcytoma (PC-12) cells (commercially available and ondeposit in the American Type Culture Collection (Deposit No. ATCC CRL1721)) are maintained in Dulbecco's modified eagles media-high glucose(DMEM-H) with 5% fetal calf serum and 5% horse serum. PC-12 cells(5×10⁵), counted by means of a hemacytometer, are plated in 25 mls of(DMEM-H) in 75 cm² untreated plastic flasks (Costar) and kept in anatmosphere of 7.5% carbon dioxide at 37° C. PC-12 cells are fed every3-4 days by decantation of the media and the addition of fresh media,and the cultures are split every week. PC-12 cells (1-5×10⁵ cells perwell) are plated in 96-well tissue culture plates and diluted inconcentrations of nerve growth factor, 2-amino-1,1,3-tricyano-1-propene,2-amino-1,1,3-tricyano-1-propene and nerve growth factor, and mediawithout additives. Neutrite outgrowth was determined, after 24, 48, and72 hours, by counting the percentage of cells with neurites that aretwice the cell body length relative to the total number of cells.Potentiation and synergism experiments are carried out with constantconcentrations of 2-amino-1,1,3-tricyano-1-propene and various nervegrowth factor concentrations over a range of 0.1 ng/ml to 100 ng/ml(subthreshold to maximum). Neurite outgrowth is determined as above.

    __________________________________________________________________________    RESULTS                                                                       Compound or Composition                                                                      Peak Conc        Count (% of cells)                            __________________________________________________________________________      nerve growth factor                                                                        50 ng/ml @ 48 hr 50                                              2-amino-1,1,3-tricyano-                                                                    20 ug/ml @ 48 hr 27                                              1-propene                                                                     nerve growth factor                                                                        0.1 ng/ml @ 48 hr                                                                                3.0                                           2-amino-1,1,3-tricyano-                                                                    132 pg/ml @ 48 hr                                                                                3.0                                           1-propene                                                                     nerve growth factor                                                                        @ 0.1 ng/ml                                                      and                      @ 48 hr                                                                            54                                              2-amino-1,1,3-tricyano-                                                                    @ 132 pg/ml                                                      1-propene                                                                     nerve growth factor                                                                        @ 0.1 ng/ml                                                      and                      @ 48 hr                                                                            50                                              2-amino-1,1,3-tricyano                                                                     @ 20 ug/ml                                                       1-propene                                                                     Control                         3.0                                         __________________________________________________________________________

Nerve growth induction, stimulation, and maintenance are achieved whenthe compositions are administered to a subject requiring such treatmentas an effective oral. parenteral, intracerebral, or intravenous dose offrom about 15 to about 45 μg/kg of body weight per day. A particularlypreferred effective amount is about 20 μg/kg of body weight per day. Itis to be understood, however, that for any particular subject, specificdosage regimens should be adjusted to the individual need and theprofessional judgment of the person administering or supervising theadministration of the aforesaid compositions. It is to be furtherunderstood that the dosages set forth herein are exemplary only and theydo not, to any extent, limit the scope or practice of the invention.

Administration of 2-amino-1,1,3-tricyano-1-propene to a host, a mammal,for example, a mouse, or a rabbit, potentiates the effects of cholineO-acetyltransferase and tyrosine hydroxylase. The potentiation of theeffects of choline-O-acetyltransferase is demonstrated as follows:

Rat adrenal pheochromocytoma (PC-12) cells (1×10⁵ per well) are platedon collagen treated 24 well plates (Costar) in dilutions of nerve growthfactor, 2-amino-1,1,3-tricyano-1-propene,2-amino-1,1,3-tricyano-1-propene and nerve growth factor, and mediawithout additives for 4 days, and choline-O-acetyl transferase activityis measured by the method of B. K. Schrier and L. Shuster, J.Neurochem., 14, 977 (1967). Briefly, after incubation media is removedfrom the plated cells and the wells are washed three times withphosphate buffer. Cells are lysed with a solution of triton X-100 andluCi of ¹⁴ C-acetyl coenzyme is added to each well. The plates are thenincubated at 37° C. for 1 hour and stopped with the addition to eachwell of 1 ml of cold water. The fluid in each well is poured over ananion exchange column, the effluent is counted by addition ofScinti-Verse E, and the activity is determined by measuringradioactivity on a scintillation counter.

    ______________________________________                                        RESULTS                                                                                              Rate of Formation                                      Compound     Conc      of Acetylcholine                                       ______________________________________                                        2-amino-1,1,3-tricyano-                                                                    132 pg/ml 11.8 ± 1.6                                                                           pmol/hr/μg of                             1-propene                        total protein                                Control                7.4 ± 0.64                                                                           pmol/hr/μg of                                                              total protein                                ______________________________________                                    

The potentiation of the effects of tyrosine hydroxylase is demonstratedas follows:

Rat adrenal pheochromocytoma (PC-12) cells (1×10⁶ cells) are plated incollagen treated 60 mm petri dishes in dilutions of nerve growth factor,2-amino-1,1,3-tricyano-1-propene, nerve growth factor plus2-amino-1,1,3-tricyano-1-propene, and media without additives, andincubated at 37° C. for 1 hour. Tyrosine hydroxylase activity ismeasured by the method of Nagatsu, et al., Analyt, Biochem., 9, 122(1964) with minor modifications. Briefly, after incubation, the platesare washed three times in phosphate buffer, scraped into 400 ul of trisacetate buffer, and frozen until assayed. For the assay, the cells arethawed, homogenized, and centrifuged. Supernatant (50 ul) is assayed fortyrosine hydroxylase activity by addition of 0.5 uCi of ³ H-tyrosine in50 ul of buffer, and incubation of the mixture at 37° C. for thirtyminutes. The reaction is terminated by addition of 200 ul of aceticacid, and activity is determined by scintillation counting of tritiatedwater contained in the effluent of the sample after treatment on ananion exchange column.

    ______________________________________                                        RESULTS                                                                       Compound   Conc       Rate of Formation of .sup.3 H.sub.2 O                   ______________________________________                                        2-amino-1,1,3-tri-                                                                       132 pg/ml  12.78 ± 0.97                                                                          fmol/hr/μg                                cyano-1-propene                  of total protein                             Control               6.25 ± 0.68                                                                           fmol/hr/μg                                                                 of total protein                             ______________________________________                                    

Choline O-acetyltransferase and tyrosine hydroxylase potentiation isachieved when the compound is administered to a subject requiring suchtreatment as an effective oral, parenteral, intracerebral, orintravenous dose of from about 15 to about 45 μg/kg of body weight perday. A particularly prefered effective amount is about 20 ∛g/kg of bodyweight per day. It is to be understood, however, that for any particularsubject, specific dosage regimens should be adjusted to the individualneed and the professional judgment of the person administering orsupervising the administration of the aforesaid compounds. It is to befurther understood that the dosages set forth herein are exemplary onlyand they do not, to any extent, limit the scope or practice of theinvention.

Effective amounts of the compound and compositions may be administeredto a subject by any one of various methods, for example, orally as incapsules or tablets, or intracerebrally, intravenously, or parenterallyin the form of sterile solutions. The compound or compositions, whileeffective themselves, may be formulated and administered in the form oftheir pharmaceutically acceptable addition salts for purposes ofstability, convenience of crystallization, increased solubility and thelike.

The compound and compositions may be administered orally, for example,with an inert diluent or with an edible carrier. They may be enclosed ingelatin capsules or compressed into tablets. For the purpose of oraltherapeutic administration, the aforesaid compound and compositions maybe incorporated with excipients and used in the form of tablets,troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums,and the like. These preparations should contain at least 0.5% of activecompound, but may be varied depending upon the particular form and mayconvenienctly be between 4.0% to about 70% of the weight of the unit.The amount of compound and compositions in such composition is such thata suitable dosage will be obtained. Prefered compositions andpreparations according to the present invention are prepared so that anoral dosage unit form contains between 1.0-300 mgs of active compound.

The tablets, pills, capsules, troches and the like may also contain thefollowing ingredients: a binder such as microcrystalline cellulose, gumtragacanth or gelatin; and excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, corn starch and thelike; a lubricant such as magnesium stearate or Sterotes; a glidant suchas colloidal silicon dioxide; and a sweetening agent such as sucrose orsaccharin or a flavoring agent such as peppermint, methyl salicylate, ororange flavoring may be added. When the dosage unit form is a capsule itmay contain, in addition to materials of the above type, a liquidcarrier such as a fatty oil. Other dosage unit forms may contain othervarious materials which modify the physical form of the dosage unit, forexample, as coatings. Thus tablets or pills may be coated with sugar,shellac, or other enteric coating agents. A syrup may contain, inaddition to the active compound or compositions, sucrose as a sweeteningagent and certain preservatives, dyes, colorings, and flavors. Materialsused in preparing these various compositions should be pharmaceuticallypure and non-toxic in the amounts used.

For the purpose of parenteral, intravenous, or intracerebral therapeuticadministration, compound and compositions may be incorporated into asolution or suspension. These preparations should contain at least 0.1%of the aforesaid compound or compositions, but may be varied between0.5% and about 50% of the weight thereof. The amount of active compoundor composition in such compositions is such that a suitable dosage willbe obtained. Prefered compositions and preparations according to thepresent invention are prepared so that a parenteral, intravenous, orintracerebral dosage unit contains between 0.5 to 100 mgs of the activecompound or composition.

The solutions or suspensions may also include the following components:a sterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parenteral,intravenous, or intracerebral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

We claim:
 1. A method of inducing nerve growth in a host requiring nervegrowth induction comprising administering a nerve growth inducingeffective amount of a composition consisting essentially of nerve growthfactor and 2-amino-1,1,3-tricyano-1-propene.
 2. A method according toclaim 1 wherein the effective amount of the composition is between about15 μg/kg of body weight and about 45 μg/kg of body weight.
 3. The methodaccording to claim 2 wherein the effective amount of the compound isabout 20 μg/kg of body weight.
 4. A method according to claim 1 whereinthe nerve is part of the central nervous system.
 5. A method accordingto claim 1 wherein the nerve is part of the peripheral nervous system.6. A method according to claim 1 wherein the nerve is part of theautonomic nervous system.
 7. The method according to claim 4 wherein thenerve of the central nervous system is selected from the groupconsisting of cholinergic and adrenergic nerves.
 8. The method accordingto claim 5 wherein the nerve of the peripheral nervous system isselected from the group consisting of the sciatic, ulnar, radial, andmedian nerves.
 9. The method according to claim 6 wherein the nerve ofthe autonomic nervous system is selected from the group consisting ofthe vagus, facial, glosso-pharyngeal, and splanchnic nerves.
 10. Themethod according to claim 1 wherein nerve growth refers to the bulk ofthe nerve.
 11. The method according to claim 1 wherein nerve growthrefers to the extension of the nerve.